本文關(guān)鍵詞：一種新的可視化環(huán)介導(dǎo)等溫擴增技術(shù)檢測瘧原蟲的研究及其應(yīng)用評價　出處：《江蘇省血吸蟲病防治研究所》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 惡性瘧原蟲 間日瘧原蟲 三日瘧原蟲 卵形瘧原蟲 可視化環(huán)介導(dǎo)等溫擴增 瘧原蟲顯微鏡檢 巢式PCR 原蟲密度

【摘要】：瘧疾是由瘧原蟲引起的嚴(yán)重危害人民健康和生命的重大傳染病，被列為全球三大公共衛(wèi)生問題之一。瘧疾也曾是嚴(yán)重危害我國人民身體健康和影響社會經(jīng)濟的重要傳染病。在上世紀(jì)60年代和70年代初，我國中部地區(qū)曾兩次暴發(fā)大范圍的瘧疾流行，在2000年前后，中部地區(qū)再次出現(xiàn)了間日瘧疫情回升和局部暴發(fā)流行。在長期的不懈努力下，我國的瘧疾防治工作取得了巨大成績，上世紀(jì)90年代在中國中部地區(qū)已成功消除了惡性瘧，到2009年，我國已有效控制了間日瘧疫情回升和局部暴發(fā)流行，瘧疾發(fā)病率已降至歷史最低水平。 我國從2010年開始實施《中國消除瘧疾行動計劃》。消除瘧疾階段的目標(biāo)、策略和措施與控制階段有很大的不同。在瘧疾控制階段，控制的目標(biāo)是降低瘧疾發(fā)病率，而在消除瘧疾階段，消除的目標(biāo)是沒有本地感染的瘧疾病例，因此，及時和準(zhǔn)確發(fā)現(xiàn)每一個可能的傳染源是阻斷瘧疾傳播的關(guān)鍵�！断懠仓笇�(dǎo)手冊》在消除瘧疾階段要求對所有疑似病例進行瘧疾實驗室檢測。但隨著瘧疾發(fā)病率的降低，臨床患者血液中原蟲密度呈下降趨勢，，低原蟲密度臨床瘧疾的比例逐年上升，給瘧疾的實驗室診斷帶來了新的挑戰(zhàn)。傳統(tǒng)的血涂片鏡檢是瘧疾實驗室診斷最常見的方法，但是鏡檢的敏感性和準(zhǔn)確性依賴于鏡檢人員的技術(shù)和經(jīng)驗，且對低原蟲密度檢測敏感性不高。新發(fā)展的瘧疾快速診斷技術(shù)（RDTs）具有簡單和快速的優(yōu)點，但現(xiàn)有RDTs對低原蟲密度感染的檢出率，尤其是對間日瘧原蟲的檢測敏感性常較低。巢式PCR等基因檢測技術(shù)具有高敏感性、高特異性的優(yōu)點，然而其需要特殊儀器和試劑，不僅價格較昂貴，且操作步驟較復(fù)雜，目前尚需要在有條件的中心實驗室開展檢測。 環(huán)介導(dǎo)等溫擴增技術(shù)（LAMP）是一種新的基因檢測技術(shù)，具有高敏感性、高特異性和操作簡便的優(yōu)勢。在具有鏈置換功能的Bst DNA聚合酶存在下，經(jīng)過一個恒溫擴增的步驟即可完成，能在短時間內(nèi)實現(xiàn)109-1010的擴增，并產(chǎn)生一種焦磷酸鎂衍生物，使反應(yīng)液呈現(xiàn)肉眼可見的白色渾濁，從而可以根據(jù)反應(yīng)液渾濁與否判定結(jié)果。在LAMP反應(yīng)體系中加入鈣黃綠素和核酸染料SYBR Green I可提高觀察效果。但鈣黃綠素敏感性不足，SYBR Green I對擴增反應(yīng)有抑制作用，需要在反應(yīng)后加入，然而由于LAMP的高效性，導(dǎo)致反應(yīng)后開蓋存在擴增產(chǎn)物污染的極大風(fēng)險。 為解決上述問題，本實驗室研發(fā)的一種針對間日瘧原蟲的可肉眼觀察結(jié)果的LAMP檢測方法，在擴增前加入含有SYBR Green I染料微晶蠟丸，并在反應(yīng)完成后可通過加熱融化釋放染料，使反應(yīng)產(chǎn)物染色并為肉眼所見。同時蠟丸在試管表面重新凝固形成屏障，可以防止反應(yīng)產(chǎn)物的污染。但該可視化LAMP技術(shù)只能檢測間日瘧原蟲感染，檢測的穩(wěn)定性也有待進一步提高。考慮到目前我國主要存在的除本地感染間日瘧病例外，外出務(wù)工人員感染輸入性惡性瘧原蟲、間日瘧原蟲、三日瘧原蟲和卵形瘧原蟲的病例也正在逐年增加，且絕大多數(shù)的瘧疾病例都發(fā)生在貧困的偏遠地區(qū)，迫切需要發(fā)展一種能同時檢測四種瘧原蟲，穩(wěn)定、敏感、特異、操作簡便、成本低廉且能夠進行大規(guī)模檢測的新技術(shù)以用于瘧原蟲的檢測。本研究在已有的可視化LAMP的基礎(chǔ)上進行改進和優(yōu)化，以應(yīng)用于對多種瘧原蟲的檢測，同時將改進的可視化LAMP試劑和市售LAMP反應(yīng)試劑盒進行成本-效果比較以評價其現(xiàn)場推廣可能性，并通過對現(xiàn)場樣本的檢測以評估其現(xiàn)場應(yīng)用價值。研究包括三個部分： 一、可視化環(huán)介導(dǎo)等溫擴增（LAMP）檢測瘧原蟲技術(shù)的改進 方法：針對瘧原蟲18S rRNA基因和線粒體基因?qū)偬禺愋员Ｊ貐^(qū)域在線設(shè)計引物，和文獻報道的引物進行比較和篩選。對反應(yīng)體系的反應(yīng)溫度、dNTPs濃度、MgSO4濃度、內(nèi)引物濃度等進行優(yōu)化。對全血和濾紙血樣本分別采用DNA提取試劑盒和簡化的加熱處理方法提取樣本DNA，評價改進的可視化LAMP技術(shù)對不同方法處理的樣本的擴增效果。以惡性瘧原蟲、間日瘧原蟲、三日瘧原蟲、卵形瘧原蟲的全血DNA作為模板來評價改進的可視化LAMP檢測瘧原蟲技術(shù)的特異性。以質(zhì)粒梯度濃度和全血樣本模擬梯度瘧原蟲密度樣本作為模板，評估改進的可視化LAMP技術(shù)的敏感性。 結(jié)果：針對瘧原蟲18S rRNA基因和線粒體基因?qū)偬禺愋员Ｊ貐^(qū)域在線設(shè)計出8對引物，經(jīng)過比較和篩選，選用文獻報道的Pg-18S rRNA引物為最適引物。經(jīng)優(yōu)化后改進的可視化LAMP的反應(yīng)體系和反應(yīng)條件為：20mM Tris-HCl pH8.8,10mM KCl,10mM (NH4)2SO4,0.1%Tween20、8U Bst DNA聚合酶、1.4mMdNTPs、9mM MgSO4、0.2μM F3和B3、2.5μM FIP和BIP、0.8μM LPF和LPB、1.0μL模板DNA，加DDW至20μL64℃反應(yīng)60min。改進的可視化LAMP檢測瘧原蟲的方法可特異地檢出四種瘧原蟲；對不同方法處理的樣本進行檢測均有較好的擴增效果；最低可檢測出3.1×100copy/μL的瘧原蟲18s rRNA基因質(zhì)粒DNA，擴增反應(yīng)的Tt值隨質(zhì)粒濃度的遞增按照相關(guān)關(guān)系遞減；對模擬的梯度瘧原蟲密度全血樣本最低可檢測出4.863p/μL的全血樣本。 二、可視化LAMP和市售LAMP試劑盒的成本-效果比較 方法：分別用自配試劑的可視化LAMP和市售的LAMP試劑盒對瘧原蟲樣本進行檢測，對兩種LAMP檢測方法的反應(yīng)效率、敏感性和特異性進行檢測效果的比較，并對兩種檢測方法的試劑成本進行比較，評價新的可視化LAMP技術(shù)用于檢測瘧原蟲的方法在現(xiàn)場推廣使用的可能性。 結(jié)果：新的可視化LAMP技術(shù)和采購的LAMP反應(yīng)試劑盒用于對瘧原蟲檢測的特異性均較高，但新的可視化LAMP擴增效率更為優(yōu)異和穩(wěn)定；優(yōu)化的可視化LAMP的檢測敏感性（4.863p/μL）比LAMP試劑盒檢測敏感性48.63p/μL高十倍；自配試劑的可視化LAMP技術(shù)檢測瘧原蟲的檢測單份樣本試劑成本約為7元/份，LAMP反應(yīng)試劑盒的成本約為166元/份，前者約為后者的1/24。 三、可視化環(huán)介導(dǎo)等溫擴增技術(shù)檢測瘧疾現(xiàn)場樣本的應(yīng)用評價 方法：采用新的可視化LAMP技術(shù)對現(xiàn)場的濾紙血樣本進行檢測，并將結(jié)果與鏡檢結(jié)果進行比較和分析，對結(jié)果不一致的樣本分別進行鏡檢復(fù)核、巢式PCR基因復(fù)核、樣本DNA濃度檢測和病例追蹤調(diào)查復(fù)核，評價新的可視化LAMP檢測瘧原蟲技術(shù)在我國消除瘧疾階段的現(xiàn)場應(yīng)用價值。 結(jié)果：采用新的可視化LAMP技術(shù)檢測臨床采集的網(wǎng)絡(luò)報告瘧疾病例濾紙血樣本200份，檢測到瘧原蟲陽性樣本186份，陰性樣本14份，與鏡檢結(jié)果的符合率為96.5%。與鏡檢結(jié)果不一致的所有樣本經(jīng)巢式PCR復(fù)核，結(jié)果與LAMP檢測結(jié)果一致。3份經(jīng)LAMP檢測和巢式PCR復(fù)核陽性，但鏡檢為陰性的樣本經(jīng)省級鏡檢專家鏡檢復(fù)核后1份為間日瘧陽性，但原蟲密度很低（16p/μL）；另2份經(jīng)鏡檢復(fù)核仍為陰性的樣本經(jīng)現(xiàn)場流行病學(xué)個案病例追蹤調(diào)查，確認均為輸入性惡性瘧病例，并在采血涂片之前均服用過抗瘧藥物；4份LAMP檢測和巢式PCR復(fù)核為陰性但鏡檢為陽性的樣本經(jīng)省級鏡檢專家鏡檢復(fù)核均為陽性，但經(jīng)樣本DNA濃度檢測發(fā)現(xiàn)沒有提取出DNA。 結(jié)論 1、本研究選用瘧原蟲屬特異性的18S rRNA引物，建立了一種可用于檢測四種瘧原蟲的可視化LAMP檢測技術(shù)，檢測敏感性達到4.863p/μL。 2、新的可視化LAMP檢測技術(shù)與市售LAMP反應(yīng)試劑盒相比，兩種方法對瘧原蟲的檢測特異性均較高，但新的可視化LAMP擴增效率和敏感性均顯著高于市售LAMP，費用僅為市售LAMP反應(yīng)試劑盒的1/24。 3、新的可視化LAMP檢測技術(shù)對現(xiàn)場采集的濾紙血樣本的檢測結(jié)果表明具有比鏡檢更高的檢測敏感性，可用于常規(guī)鏡檢很難檢測的低原蟲密度和已服用過抗瘧藥物患者血樣的檢測。 4、新的可視化LAMP檢測技術(shù)具有敏感、高效、簡便、快捷和可批量檢測的優(yōu)點，可作為瘧原蟲基因檢測的一個新工具，在我國消除瘧疾階段中推廣應(yīng)用。
[Abstract]:Malaria is caused by parasites seriously endanger people's health and life of the major infectious diseases, is listed as one of the world's three major public health problems. Malaria was also a serious harm to the health of our people and the social and economic impact of infectious diseases. In the last century and the beginning of 70s 60s, the malaria epidemic in the middle area of China was two in a large range, before and after 2000, the central region again rebounded vivax malaria epidemic and outbreak. In the long-term unremitting efforts, the work of malaria prevention and control in China has made great achievements, in 90s in the central region of China has successfully eliminated the pernicious malaria, by 2009, China has been effectively controlled the rise of vivax malaria epidemic and outbreak, malaria incidence rate has dropped to the lowest level in history.
China began to implement the "Chinese action plans to eliminate malaria. Malaria elimination goals from 2010, strategies and measures and control stage are very different. In the stage of malaria control, the control objective is to reduce the incidence of malaria, and in the elimination of malaria elimination target stage, there is no local infection of malaria cases, therefore, timely and accurately find every possible source of infection is the key to blocking the spread of malaria. Malaria elimination guide in malaria elimination stage > requirements for all suspected cases of malaria in laboratory. But with the malaria incidence decreased in patients with clinical blood parasite density decreased, low proportion of clinical malaria parasite density rise, bring new challenges to the laboratory diagnosis of malaria blood smear. The traditional is the most common method for laboratory diagnosis of malaria, but the sensitivity and accuracy of examination Examiners rely on technology and experience, and the low parasite density detection sensitivity is not high. The development of new technology for rapid diagnosis of malaria (RDTs) has the advantages of simple and fast, but the existing RDTs detection rate of low parasite density infection, especially the detection sensitivity of Plasmodium vivax nested PCR is often low. Gene detection technology has the advantages of high Gao Min sensibility, specificity, but it requires special equipment and reagents, not only the price is more expensive, and the operation steps are complex, it is necessary to carry out detection in central laboratory conditions.
Loop mediated isothermal amplification (LAMP) is a new genetic test, Gao Min has high specificity and sensitivity, simple operation and advantages. In the presence of with strand displacement function of Bst DNA polymerase, after an isothermal amplification step can be completed in a short period of time, can realize the amplification of 109-1010. And produce a kind of magnesium pyrophosphate derivatives, the reaction liquid showed visible white haze, according to reaction liquid turbidity and determining result. In LAMP reaction with calcein and Green nucleic acid dye SYBR I can improve the observation effect. But the lack of sensitivity of calcein, SYBR Green I has inhibitory effect on need to join in the amplification reaction, after the reaction, however, the efficiency of LAMP, leading to a significant risk response after opening the cover are amplified product contamination.
To solve the above problems, according to a kind of visual observation results can vivax LAMP detection method developed by our lab, before joining Green SYBR in amplification of I dye containing microcrystalline wax pill, and after the completion of the reaction can release the dye by heating to melt, the reaction product was seen to the naked eye. At the same time in the test tube to the surface of Lawan the formation of solidification barrier, reaction products can prevent pollution. But only LAMP the visual detection of Plasmodium vivax infection, detection stability also needs to be further improved. Considering the infection of Plasmodium vivax malaria cases in China are mainly imported, out of Plasmodium falciparum, Plasmodium vivax infection of migrant workers, three of Plasmodium vivax and oval Plasmodium cases are increasing year by year, and the vast majority of malaria cases have occurred in the remote areas of poverty, but also an urgent need to develop Detection of four species of Plasmodium, stable, sensitive, specific, simple operation, low cost and new technology to be able to carry out large-scale detection for malaria detection. This study was improved and optimized based on the visualization of LAMP, applied to a variety of parasite detection, at the same time will improve the visualization of LAMP reagent and city the sale of LAMP reagent kit cost-effectiveness to evaluate the possibility of site promotion, and through the test to the sample to evaluate its application value. The research includes three parts:
Improvement of Plasmodium technique by visual loop mediated isothermal amplification (LAMP)
Methods: according to the 18S rRNA gene and mitochondrial gene of Plasmodium genus specific conserved region online design primers, primers and reported in the literature were selected and compared. The reaction temperature, the reaction system of dNTPs concentration, MgSO4 concentration, inner primer concentration were optimized. The blood and filter paper blood samples were extracted by DNA Kit and simplified the heat treatment method of sample DNA extraction, amplification effect visualization technology assessment of LAMP improved treatment on different methods of sample. With Plasmodium falciparum, Plasmodium vivax, three Plasmodium vivax, specific Plasmodium falciparum blood DNA as template to evaluate the visualization technology of LAMP detection of Plasmodium is improved. The plasmid concentration gradient and blood sample simulation gradient the parasite density sample as the template, sensitivity of visual LAMP technology improved evaluation.
Results: the 18S rRNA gene and mitochondrial gene of Plasmodium genus specific conserved region of online designed 8 pairs of primers, through comparison and selection, selection of Pg-18S rRNA primers reported in the literature is the most suitable primers. The reaction system and the reaction conditions have been optimized to improve the visualization of LAMP is: 20mM Tris-HCl pH8.8,10mM KCl 10mM (NH4) 2SO4,0.1%Tween20,8U Bst DNA 1.4mMdNTPs, 9mM MgSO4,0.2 polymerase, M F3 and B3,2.5 M and FIP BIP, 0.8 M LPF and 1 L LPB, DNA template, LAMP and visual detection of Plasmodium DDW to 20 DEG C L64 60min. improved the reaction assay were detected in four different Plasmodium; different methods of processing samples have good amplification effect; the minimum detectable Plasmodium 18S rRNA gene plasmid DNA 3.1 * 100copy/ L, amplification of Tt with the increasing concentration of the plasmid in accordance with the relevant relation of decline; simulation The whole blood sample of the density Plasmodium Plasmodium density can detect the whole blood samples of 4.863p/ mu L.
Two, the cost effectiveness comparison of the visual LAMP and the marketed LAMP Kit
Methods: to detect the parasite samples by LAMP kit and LAMP visualization of commercially available self prepared reagents, the reaction efficiency of two kinds of LAMP detection methods, compare the detection results of sensitivity and specificity, and the reagent cost of two detection methods were compared on visual LAMP technology a new method for price the detection of Plasmodium falciparum in possibility to promote the use of the site.
Results: LAMP reaction kit new visual LAMP technology and procurement for high specificity for detection of Plasmodium, but new visual LAMP amplification efficiency is more excellent and stable; the sensitivity of visualization optimization of LAMP (4.863p/ L) ten times higher than the sensitivity of the 48.63p/ detection kit of LAMP L; visualization LAMP detection of Plasmodium homemade reagent detection of single sample reagent cost is about 7 yuan / share, LAMP reaction kit costs about 166 yuan / share, the former is about 1/24. of the latter
Three, application evaluation of visual loop mediated isothermal amplification for detection of malaria site samples
Methods: the blood samples were detected by filter paper LAMP visualization technology, and the results of the examination results were compared and analyzed, the inconsistent results were microscopic review, review of PCR gene nested DNA concentration detection, sample survey and case review, evaluation of new technology of visual detection of Plasmodium LAMP the application value of malaria elimination stage in our country.
Results: using the new detection technology of visualization LAMP clinical acquisition network reported malaria cases filter paper blood samples of 200, detection of Plasmodium positive of 186 samples, 14 samples were negative, and the microscopic examination results were consistent with all samples with 96.5%. and microscopic examination results by nested PCR review results and test results of LAMP a consistent.3 detected by LAMP and nested PCR positive review, but microscopic examination for negative samples by the provincial examination expert examination review after 1 for vivax malaria positive, but parasite density is very low (16p/ L); the other 2 were by the microscopic examination review still negative samples by means of epidemiological case tracking the investigation, confirmed imported cases of falciparum malaria, and in blood smear before were treated with anti malarial drugs; 4 LAMP detection and nested PCR review was negative but positive samples for microscopic examination by the provincial examination examination review experts were positive, but the kind of DNA concentration detection found no extraction of DNA.
conclusion
1, in this study, Plasmodium specific 18S rRNA primers were selected to establish a visualized LAMP detection technique for detecting four Plasmodium species, and the detection sensitivity reached 4.863p/ L..
2, compared with the commercially available LAMP reaction kit, the two new methods for detection of Plasmodium falciparum were all highly specific, but the efficiency and sensitivity of the new visualization LAMP amplification were significantly higher than those of the commercially available LAMP LAMP. The cost was only 1/24. of the commercially available LAMP reaction kit.
3, the new visual LAMP detection technology shows that the detection results of filter paper blood samples collected at the scene show higher sensitivity than microscopy. It can be used for the detection of low protozoan density and the blood samples taken from antimalarial drugs which are difficult to detect by routine microscopy.
4, the new visualization LAMP detection technology has the advantages of sensitivity, efficiency, simplicity, rapidness and mass detection. It can be used as a new tool for gene detection of Plasmodium, and it is widely applied in the elimination stage of malaria in China.

【學(xué)位授予單位】：江蘇省血吸蟲病防治研究所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R440;R382.31

【參考文獻】

相關(guān)期刊論文 前7條

1 蘆春斌;羅樂;楊夢婕;聶凱;王淼;馬學(xué)軍;;基于顏色判定的環(huán)介導(dǎo)等溫擴增技術(shù)檢測HPV6和HPV16[J];病毒學(xué)報;2011年01期

2 朱韓武;曹俊;周華云;李菊林;朱國鼎;顧亞萍;王偉明;劉耀寶;陶志勇;高琪;;環(huán)介導(dǎo)等溫擴增技術(shù)檢測蚊體內(nèi)間日瘧原蟲子孢子的研究[J];中國血吸蟲病防治雜志;2010年02期

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4 高琪,尚樂園,顧政誠;我國中部地區(qū)目前的瘧疾形勢[J];中國寄生蟲病防治雜志;2002年04期

5 楊秋林;許麗芳;張愉快;王可耕;;環(huán)介導(dǎo)等溫擴增技術(shù)檢測間日瘧原蟲的研究[J];中國病原生物學(xué)雜志;2008年08期

6 周水森;王漪;房文;湯林華;;2007年全國瘧疾形勢[J];中國寄生蟲學(xué)與寄生蟲病雜志;2008年06期

7 周水森;王漪;房文;湯林華;;2008年全國瘧疾形勢[J];中國寄生蟲學(xué)與寄生蟲病雜志;2009年06期

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